PROJECT SUMMARY The epididymis provides a unique luminal fluid microenvironment that allows for sperm maturation and survival, and disruptions to this function lead to male infertility. Further, disruptions to epididymal function may also arise as a consequence of abnormal fetal development, although very little is known either of the process of Wolffian duct/epididymal development or of the nature and causes of congenital defects that lead to male infertility. The central hypothesis of this application is that elongation and coiling of the Wolffian duct is crucial for the function of the resulting epididymis, and that failure to elongate and coil leads to male infertility. The overall goal is two-fold, to examine: (a) the mechanisms by which cells move to elongate the duct and (b) to dissect the underlying cellular and molecular mechanisms that regulate those cell movements. Although we have evidence that epithelial cells move by intercalating, we will test the hypothesis that intercalation-type movements of the mesenchyme cells that surround the duct and the en masse movement of mesenchymal cells in the interstitium contribute to ductal elongation and coiling. A combination of genetically modified mice, advanced microscopy including confocal, second harmonic two-photon, and atomic force microscopy, in vitro organ culture and contemporary software analyses will be used to test the hypotheses outlined in the following three specific aims: (1) To test the hypothesis that the stiffness (modulus) of the ECM undergoes dynamic changes during Wolffian duct morphogenesis thereby providing a biomechanical environment that promotes epithelial and mesenchymal cell intercalation and en masse movement of mesenchymal cells within the interstitium. (2) To test the hypothesis that radial intercalation of mesenchymal cells and the en mass movements of mesenchymal cells within the interstitium are major drivers of Wolffian duct elongation and coiling. (3) To test the hypothesis that Ptk7 and Rac1 regulate radial intercalation and en masse movement of mesenchymal cells via regulating ECM biomechanical properties through changes in its deposition and assembly during Wolffian duct development, which in turn are important for male fertility. Therefore, this application focuses on the role of the ECM during Wolffian duct morphogenesis paying special attention to the importance of its assembly, distribution and stiffness, and how this is coordinated to regulate mediolateral and radial intercalation of epithelial and mesenchymal cells respectively, and the en masse movement of mesenchymal cells within the interstitium. Coordination of these events is critical for Wolffian/epididymal duct development, and therefore, male fertility. The anticipated outcomes of this study will not only have a major impact on an area of reproductive biology that has been poorly understood, but will also contribute to our understanding of the fundamental process of tubular morphogenesis. Specifically they will provide an understanding as to how the regulation of growth of the epididymis during development is important clinically.